Fluorescent N-alkylated acrylamide copolymers and optical pH sensors

ABSTRACT

The invention relates to a polymer composition comprising pH-sensitive fluorescent dyes, to an ionic strength-independent optical sensor for pH value determination that contains the composition in the form of a membrane on a transparent support material, and to an optical process, according to the fluorescence method, that renders possible highly accurate pH value determination independently of the ionic strength of the test solution. The process is especially suitable for the determination of the pH value of physiological solutions, especially for the determination of the pH value of blood.

[0001] The invention relates to a polymer composition comprisingpH-sensitive fluorescent dyes, to an ionic strength-independent opticalsensor for pH value determination that contains the composition in theform of a membrane on a transparent support material, and also to anoptical process, according to the fluorescence method, that renderspossible highly accurate pH value determination independently of theionic strength of the test solution. The process is especially suitablefor the determination of the pH value of physiological solutions,especially for the determination of the pH value of blood.

[0002] It is known that the pK_(a) value of an indicator varies with theionic strength of a solution and that that variation depends on thelevel of the charge at the indicator. For example, it has already beenproposed in DE-A-3 430 935 to determine computationally the ionicstrength and the pH value from the difference between the measuredvalues of two sensors having different ionic strength dependence ofwhich one exhibits as low as possible an ionic strength dependence,after calibration of said sensors with known test solutions. The sensordescribed therein that is almost independent of the ionic strength doesnot lie exactly within the physiological pH range and has a lowresolution. The construction of those sensors is effected withoutembedding into a polymer matrix and consequently has the disadvantagethat the dye is in direct contact with the test solution. Thefluorescent dye of the sensors, which is the same in each case, is inthat arrangement immobilised directly on the surface of glass supportsby way of bridging groups, one of the sensors containing additionalcharges for achieving a high polarity and ionic strength dependence andthe other sensor being so modified that it is essentially non-polar,hydrophobic and independent of the ionic strength. A quite considerabledisadvantage of those sensors is that the fluorescent dye is exposeddirectly to external influences of the test solutions, and both physicalinfluences (for example dissolution of the dye, deposits on the surface)and chemical influences (decomposition of the dye) quickly make thesensors unusable. In addition, in the case of excitations in anevanescent field, interference between the evanescent measuring fieldand the fluorescence of the test sample cannot be completely avoided,which reduces the accuracy of the measurement. The response time ofthose sensors is on the other hand short, since the fluorescent dyebonded to the surface immediately comes into contact with the testsolution. The sensitivity is regarded as adequate.

[0003] The method of optical pH determination using two sensors thatrespond to different extents to the ionic strength of a test solution isexpensive in respect of apparatus and a subsequent, additionalcalculation step has to be carried out.

[0004] It has now been found that, by selection of quite specificcopolymers of acrylamides and methacrylamides in conjunction with theselection of a narrow concentration range of a fluorescent dye, which isembedded in the polymer matrix, it is possible to produce an optical pHsensor that allows highly accurate optical pH measurement that isindependent of ionic strength in the physiological pH range of from 6.5to 8.2. By that means, a second measurement and the calculation step foreliminating the ionic strength are dispensed with. The high degree ofaccuracy of the pH value measurement is of great importance especiallyin the analysis of human blood, since the measurement can be used, forexample, for monitoring the therapy of metabolic diseases. For a quickand inexpensive test it is therefore especially advantageous if only onesensor has to be used. The analytical apparatus can consequently also beminiaturised more easily.

[0005] The shelf life and working life of those sensors is high sincethe fluorescent dye is effectively protected by the polymer matrixagainst damaging or interfering influences of the test medium. Thesensitivity is not reduced in such sensors and the response times aresurprisingly short.

[0006] By means of the polymer compositions it is possible to set veryaccurately, for example, the hydrophilic property, hydrophobic property,polarity and/or dielectric constant of the matrix, which, combined withthe selected concentration range of the fluorophore, results in ameasurement that is independent of ionic strength within a particular pHvalue range.

[0007] The response times and the conditioning times correspond to theshort periods of time required of optical measuring systems despiteembedding of the fluorophore, those parameters being dependentessentially on the membrane thickness.

[0008] The invention relates to water-insoluble copolymers that arecomposed of

[0009] a) from 39.9 to 60% by weight of N,N-dimethylacrylamide orN,N-dimethylmethacrylamide;

[0010] b) from 60 to 39.9% by weight of a monomer of formula Ia or Ib

[0011]  wherein R_(a) is hydrogen or C₁-C₆alkyl and R_(b) isC₁-C₁₂alkyl; with the proviso that R_(a) and Rb are not both methyl;

[0012] c) from 0.1 to 0.7% by weight of a proton-sensitive fluorophorewhich is covalently bonded to the polymer; and

[0013] d) from 0 to 20% by weight of a diolefinic crosslinkingcomponent, the sum of the percentage weights of a) to d) being 100%.

[0014] Within the scope of the present invention, “water-insoluble”denotes that at most traces of less than 0.1% are able to dissolve. Inorder, on the other hand, to be able to produce a good contact with thetest medium, the copolymer must, however, be swellable.

[0015] The alkyl radicals may be linear or branched. Examples ofC₁-C₁₂alkyl are the linear or branched radicals: methyl, ethyl and thevarious position isomers of propyl, butyl, pentyl, hexyl, heptyl, octyl,nonyl, decyl, undecyl or dodecyl.

[0016] The monomer preferably used as monomer a) isN,N-dimethylacrylamide. Preferred water-insoluble copolymers areobtained when R_(a) is hydrogen and R_(b) is a branched C₃-C8alkyl.Especially preferred are water-insoluble copolymers in which R_(a) ishydrogen, R_(b) is tertiary butyl and the ratio of monomer a) to monomerb) is 50 parts by weight to 50 parts by weight.

[0017] Another group of preferred water-insoluble copolymers is obtainedwhen R_(a) is methyl or ethyl and R_(b) is linear C₃-C₈alkyl. Especiallypreferably, R_(a) is methyl and R_(b) is n-butyl.

[0018] Suitable proton-sensitive fluorescent dyes are, for example,those from the group of the xanthenes and benzoxanthenes, for examplefluorescein, halogenated fluoresceins, seminaphthofluoresceins,seminaphthorhodafluors, 2,3-benzo fluorescein, 3,4-benzofluorescein, theisomers of benzorhodamine and substituted derivatives, the isomers ofbenzochromogen and substituted derivatives; acridines, for exampleacridine, 9-amino-6-chloroacridine; acridones, for example7-hydroxyacridone and 7-hydroxybenz acridone; pyrenes, for example8-hydroxypyrene-1,3,6-trisulfonic acid; cyanine dyes; and coumarins, forexample 7-hydroxycoumarin and 4-chloromethyl-7-hydroxycoumarin. Thefluorescent dyes may be functionalised with olefinically unsaturatedgroups in order to bind to the polymer backbone.

[0019] Preferably, the fluorophores are selected from the groupconsisting of acridines, acridones, rhodamines, xanthenes,benzoxanthenes, pyrenes and coumarins, which are either admixed with orcovalently bonded to the polymer.

[0020] Preferred are water-insoluble copolymers in which the flourophoreis covalently bonded to the polymer.

[0021] Especially preferred are water-insoluble copolymers in which thefluorophore is a compound of formula II, III, IV, V or VI

[0022] wherein

[0023] R₁, R₂, R₅ and R₆ are each independently of the others hydrogen,—SO₂—(C₁-C₆)alkylphenyl, C₁-C₃₀alkyl, C₁-C₃₀alkyl-CO— or a radical ofthe formula —(C_(n)H_(2n)—O—)_(m)—R₈;

[0024] R₃ is hydrogen or —SO₂—(C₁-C₆)alkylphenyl;

[0025] R₄and R₇ are a C₁-C₃₀alkylene or a radical of the formula—(C_(n)H_(2n)—O—)_(m)—R₈;

[0026] Z is a divalent radical —NH—CO—;

[0027] R₈ is a direct bond or C₁-C₁₂alkylene;

[0028] n is an integer from 2 to 6 and m is an integer from 1 to 10,with the proviso that the total number of carbon atoms is no more than30;

[0029] R₉ and R₁₀ are each independently of the other H, C₁-C₄alkyl,C₁-C₄alkoxy, C₁-C₄alkoxycarbonyl, C₁-C₄alkyl-SO₂— or halogen, and either

[0030] R₁₁ is H and R₁₂ is a divalent radical —NH—CO—,—CO—NH—(C₂-C₁₂alkylene-O)—CO—, — CO—NH— (C₂-C₁₂alkylene-NH)—CO— or—C(O)—NH—(CH₂CH₂—O)_(1 to 6)—CH₂C(O)— NH—, or R₁₁ is a divalent radical—NH—CO—, —CO—NH—(C₂-C₁₂alkylene-O)—CO—, —CO—NH—(C₂-C₁₂alkylene-NH)— CO—or —C(O)—NH—(CH₂CH₂—O)_(1 to 6)—CH₂C(O)—NH—, and R₁₂ is H; or whereineither

[0031] R₁₃ is H and R₁₄ is a divalent radical —NH—C(O)—,—CO—NH—(C₂-C₁₂alkylene-O)—CO—, — CO—NH— (C₂-C₁₂alkylene-NH)—CO— or—C(O)—NH—(CH₂CH₂—O)_(1 to 6)—CH₂C(O)— NH—, or R₁₃ is a divalent radical—NH—C(O)—, —CO—NH—(C₂-C₁₂alkylene-O)—CO—, —CO—NH—(C₂-C₁₂alkylene—NH)—CO— or —C(O)—NH—(CH₂CH₂—O)_(1 to 6)—CH₂C(O)—NH—, and R₁₄ is H,wherein the radical — COOH is each in free form or in salt form, or aC₁-C₂₀alkyl ester thereof.

[0032] Alkyl as such or as a structural element of other groups, suchas, for example, of alkoxy and alkoxycarbonyl is, with appropriateconsideration given in each case to the number of carbon atomsrespectively included in the corresponding group or compound, eitherstraight-chain, that is to say methyl, ethyl, propyl or butyl, orbranched, e.g. isopropyl, isobutyl, sec-butyl or tert-butyl.

[0033] Halogen is fluorine, chlorine, bromine or iodine, especiallyfluorine, chlorine or bromine, more especially chlorine or bromine.

[0034] Examples from which the divalent radicals Z, R₁₁, R₁₂, R₁₃ andR₁₄ may arise are the acryloylamine group —NHCOCH═CH₂, themethacryloylamine group —NHCOC(CH₃)═CH₂, and the2-(methacryloyloxy)-ethylaminocarbonyl group —CONHCH₂CH₂OCOC(CH₃)═CH₂.

[0035] Preferably, R₁ and R₂ of the fluorophore of formula II are eachindependently of the other hydrogen or linear C₁₂-C₂₄alkyl.

[0036] Also preferably, R₄ of the fluorophore of formula II is a linearC₂-C₁₆alkylene or a radical of the formula —(C₂H₄—O—)_(m)—R₈ wherein R₈and m are as defined hereinbefore.

[0037] Another group of preferred water-insoluble copolymers is formedby those in which R₅ and R₆ of the fluorophore of formula III are eachindependently of the other linear C₂-C₁₂alkyl.

[0038] Copolymerisable fluorescent dyes contain, for example, anethylenically unsaturated group (vinyl, crotonyl, methallyl) that isbonded directly or via a bridging group to the fluorescent dye. Themonomers a) and b) are known. A known copolymerisable fluorescent dyeis, for example, 3-oder 4-acryloylaminofluorescein.

[0039] Polymers with fluorescent dyes comprising the bridging groups—O—C(O)— and —C(O)—O— C₂-C₁₂alkylene—O—C(O)— are obtainable, forexample, by esterification with fluorescent dyes that contain carboxylor hydroxyl groups. Polymers with fluorescent dyes comprising thebridging groups —NH—C(O)—O— and —NH—C(O)—O—C₂-C₁₂alkylene—O—C(O)— areaccessible, for example, by way of isocyanate-functionalised fluorescentdyes and hydroxyl group-containing polymers.

[0040] The reactions described above may be carried out in a mannerknown per se, for example in the absence or presence of a suitablesolvent, as required with cooling, at room temperature or with heating,e.g. in a temperature range of from approximately 5° C. to approximately200° C., preferably approximately from 20° C. to 120° C., and, ifnecessary, in a closed vessel, under pressure, in an inert gasatmosphere and/or under anhydrous conditions.

[0041] The preparation of the polymers may be carried out according tomethods known per se.

[0042] The reactants may be reacted with one another as they are, thatis to say without the addition of a solvent or diluent, e.g. in themelt. Generally, however, the addition of a solvent or diluent or of amixture of solvents is advantageous. There may be mentioned as examplesof such solvents and diluents: water; esters, such as ethyl acetate;ethers, such as diethyl ether, dipropyl ether, diisopropyl ether,dibutyl ether, tert-butyl methyl ether, ethylene glycol monomethylether, ethylene glycol monoethyl ether, ethylene glycol dimethyl ether,dimethoxy diethyl ether, tetrahydrofuran or dioxane; ketones, such asacetone, methyl ethyl ketone or methyl isobutyl ketone; alcohols, suchas methanol, ethanol, propanol, isopropanol, butanol, ethylene glycol orglycerol; amides, such as N,N-dimethylformamide, N,N-diethylformamide,N,N-dimethylacetamide, N-methylpyrrolidone or hexamethylphosphoric acidtriamide; nitrites, such as acetonitrile or propionitrile; andsulfoxides, such as dimethyl sulfoxide.

[0043] The copolymerisable fluorescent dyes may be prepared according toprocesses known per se, and the starting materials are either availablecommercially or can be prepared according to analogous processes.

[0044] One possible method of preparing compounds of formula II or IIIcomprises

[0045] a) in compounds of formula IIc or IIIc

[0046]  removing the phthalimide group under acidic conditions and,where appropriate, in a second step

[0047] b) further reacting the reaction products with acrylic acidchloride or methacrylic acid chloride or

[0048] c) where appropriate, removing the para-toluenesulfonyl groupfrom the reaction products of the starting materials of formula IIcunder acidic conditions,

[0049] the radicals R₁, R₂, R₄, R₅, R₆ and R₇ being as definedhereinbefore.

[0050] The methods for the removal of the protecting groups are knownper se and may be used in an analogous manner in the preparation of thecompounds of formulae II and III.

[0051] The compounds of formula IIc can be prepared in a manner knownper se by stepwise alkylation with different alkylating agents, oralkylation with an alkylating agent or acylating agent of commerciallyavailable 3,6-diaminoacridine. Suitable alkylating agents are, forexample, dialkyl sulfates or monohaloalkanes, especially chloro-, bromo-and iodo-alkanes. Suitable acylating agents are, for example, carboxylicacid anhydrides and, especially, carboxylic acid halides, such as, forexample, carboxylic acid chlorides. That reaction may be carried out inthe presence of inert polar and aprotic solvents, for example ethers,alkylated acid amides and lactams or sulfones, and at elevatedtemperatures, for example from 50 to 150° C. Expediently, a hydrogenhalide acceptor is added, for example an alkali metal carbonate or atertiary amine, especially a sterically hindered tertiary amine.

[0052] The compounds of formula IIIc are obtainable, for example, by thereaction of phthalic acid anhydride with 2 molar equivalents of3-monoalkylaminophenol. Another possible method of preparation is thereaction of 3-monoalkylaminophenol with one molar equivalent of2-hydroxy-4-dialkylamino-2′-carboxyl-benzophenone. Those reactions aredescribed, for example, in U.S. Pat. No. 4,622,400.

[0053] Compounds of formulae IV, V and VI may be prepared in ananalogous manner.

[0054] Conditions for the reactions are known per se. They may becarried out, for example, in the presence of a suitable solvent ordiluent or a mixture thereof, as required with cooling, at roomtemperature or with heating, e.g. in a temperature range of fromapproximately −10° C. to the boiling temperature of the reactionmixture, preferably from approximately 0° C. to approximately 25° C.,and, if necessary, in a closed vessel, under pressure, in an inert gasatmosphere and/or under anhydrous conditions. Especially advantageousreaction conditions are disclosed in the Examples.

[0055] Preferably, the copolymers have a mean molecular weight of from 2000 to 500 000, especially from 10 000 to 350 000 daltons, determinedaccording to the gel permeation method using standard polymers of knownmolecular weight.

[0056] The water-insoluble copolymers may be crosslinked in the form ofa layer, for example with from 0.01 to 20%, preferably from 0.1 to 10%,and especially preferably from 0.5 to 5%, by weight of a crosslinkingagent based on the polymer. Suitable crosslinking agents are, forexample, acrylic acid or methacrylic acid esters or amides of polyols,preferably diols to tetrols, or polyamines, preferably diamines totetramines. Such crosslinking agents are known and widely described inthe literature. Some examples of polyols are ethylene glycol,1,3-propanediol, 1,4-butanediol, 1,6-hexanediol, diethylene glycol,1,1,1-trihydroxymethyl ethane or propane, pentaerythritol anddipentaerythritol. Some examples of polyamines are ethylenediamine,1,3-propanediamine, 1,4-butanediamine, 1,6-hexanediamine,diethylenetriamine and triethylenetetramine. Another known crosslinkingagent is, for example, divinylbenzene. Alkylene-bis-dialkylmaleimidylcompounds, for example, are also suitable, for exampleethylene-bis-(dimethyl)maleimidyl.

[0057] The invention relates also to a composition and to an opticalsensor for an ionic strength-independent determination of pH value,consisting of

[0058] A) a transparent support material

[0059] B) a layer of water-insoluble copolymers that are composed of

[0060] a) from 39.9 to 60% by weight of N,N-dimethylacrylamide orN,N-dimethylmethacrylamide;

[0061] b) from 60 to 39.9% by weight of a monomer of formula Ia or Ib

[0062]  wherein R_(a) is hydrogen or C₁-C₆alkyl and R_(b) isC₁-C₁₂alkyl; with the proviso that R_(a) and Rb are not both methyl;

[0063] c) from 0.1 to 0.7% by weight of a proton-sensitive fluorophorewhich is covalently bonded to the polymer; and

[0064] d) from 0 to 20% by weight of a diolefinic crosslinkingcomponent,

[0065] the sum of the percentage weights of a) to d) being 100%.

[0066] The copolymer and fluorescent dye preferences given above applylikewise to the sensor.

[0067] Sensors in which the fluorophore is admixed with the polymer aresuitable principally for once-only use. If the polymer membrane isprovided with a permeable and hydrophilic protective layer, then boththose sensors and also, in general, sensors with polymer-bondedfluorophores, which may also contain a protective layer on the membrane,may be used repeatedly or for continuous measurements.

[0068] The geometric form of the support material may vary widely; itmay be, for example, fibres, cylinders, spheres, cuboids or cubes.Furthermore, flow systems in which continuous measurements or successivemeasurements may be carried out are possible. Planar sensors arepreferred. The support material is transparent. It may be, for example,inorganic glass or transparent plastics, such as a polycarbonate, apolyester (for example polyethylene terephthalate), a polyamide, or apolyacrylate or polymethacrylate.

[0069] The planar sensor may be of any external shape, for examplesquare, rectangular or round. It may have a surface area of from 0.01 toapproximately 50 cm², more advantageously from 0.02 to 10 cm². Themeasuring zone of the sensor may have a surface area of less than 5 mm²,preferably less than or equal to 2 mm². The measuring zone may becorrespond exactly to one completely coated surface of the sensor.Advantageously, a coating that is on both sides but that is locallyseparated may be used.

[0070] The sensor may comprise one or more locally separated membranelayers; in the latter case parallel measurements may be carried out withidentical or different test samples.

[0071] Preferably, the thickness of the polymer layer B) is from 0.1 to500 μm, especially preferably from 1 to 100 μm.

[0072] The production of such layers can be carried out in a mannerknown per se, for example by dissolving the composition in an organicsolvent, then casting to form a film and finally removing the solvent.

[0073] Also possible for the production of the layers are processesknown from coating technology. Examples are spin-coating, spraying orknife application processes, with spin-casting processes beingpreferred.

[0074] Suitable solvents include alcohols, ethers, esters, acid amidesand ketones. Especially suitable are readily volatile solvents,especially tetrahydrofuran.

[0075] In addition to those processes, in which the composition is firstof all dissolved, moulded and the solvent subsequently evaporated again,hot-moulding processes are also possible, since the composition is athermoplastic material. Suitable processes include extrusion, injectionmoulding, pressing or blowing processes as known from thermoplasticplastics processing.

[0076] The layer may be transparent or slightly opaque. Preferably it istransparent.

[0077] In order to improve the adhesion, the support materials may betreated beforehand with adhesion promoters. For the same purpose, aplasma treatment of the support material in order to produce functionalgroups on the surface is also possible. The surface may also be providedwith copolymerisable groups in order to achieve an especially high levelof adhesion. Known adhesion promoters for glasses are, for example,triethoxy-glycidyloxy-silane, 3-azidopropyl-triethoxysilane and3-aminopropyl-triethoxysilane. The thus treated surfaces may be furthermodified, for example withO—(N-succinimidyl)-6-(4′-azido-2′-nitrophenylamino)-hexanoate.

[0078] The invention relates also to a process for the ionicstrength-independent, reversible optical determination of the pH valueof an aqueous sample according to the fluorescence method, in whichprocess an optical sensor, consisting of

[0079] A) a transparent support material

[0080] B) a layer of water-insoluble copolymers that are composed of

[0081] a) from 39.9 to 60% by weight of N,N-dimethylacrylamide orN,N-dimethylmethacrylamide;

[0082] b) from 60 to 39.9% by weight of a monomer of formula Ia or Ib

[0083]  wherein R_(a) is hydrogen or C₁-C₆alkyl and R_(b) isC₁-C₁₂alkyl; with the proviso that R_(a) and Rb are not both methyl;

[0084] c) from 0.1 to 0.7% by weight of a proton-sensitive fluorophorewhich is covalently bonded to the polymer; and

[0085] d) from 0 to 20% by weight of a diolefinic crosslinkingcomponent,

[0086] the sum of the percentage weights of a) to d) being 100%, isbrought into contact with an aqueous test sample and irradiated withexcitation light, the fluorescence is measured, and the pH value iscalculated from the measured fluorescence intensity taking calibrationcurves into consideration.

[0087] The above-described preferences in respect of the copolymers andfluorescent dyes apply likewise to the sensor.

[0088] In detail, the procedure may be as follows: after calibrationwith samples of known pH, a measurement of the fluorescence intensity incontact with a test solution of unknown composition is carried out andthe pH with respect to the measured fluorescence intensity is determineddirectly from the calibration.

[0089] The sensors are brought into contact with the calibratingsolutions and with the test samples. This may be effected by hand (forexample by means of pipetting) or using a suitable automatic flowsystem, the sensors being mounted in fixed position in a flow cell. Suchflow cells are known to the person skilled in the art and may be adaptedin a simple manner to the purpose in question.

[0090] UV lamps (for example mercury vapour lamps, halogen lamps),lasers, diode lasers and light-emitting diodes may be used as lightsources for the excitation of the fluorescence. It may be expedient tofilter out, by means of filters, light of the wavelength at which thefluorescent dye has an absorption maximum. The fluorescent light emittedby the sensors can be collected, for example using a lens system, andthen directed to a detector, for example a secondary electron multiplieror a photodiode. The lens system may be so arranged that thefluorescence radiation is measured through the transparent support, viathe edges of the support, or via the analytical sample. Advantageously,the radiation is deflected in a manner known per se by means of adichroic mirror. The fluorescence of the sensors is measured preferablyduring contact with the calibrating solutions or sample solutions.

[0091] The measurement may be effected under photostationary conditionswith continuous illumination, but can, if required, alternatively betime-resolved. This can be achieved, for example, by a laser pulse oflimited duration or by modulation of the intensity of a light source.

[0092] The response times may be less than 30 seconds and a firstmeasurement is already possible after less than about 5 minutes. Thesensors are furthermore distinguished by a high storage stability.

[0093] Preferably, the process is used for test solutions that have a pHof from 6.5 to 8.5, especially preferably a pH value of from 6.7 to 7.8.

[0094] The ionic strength of the test solution is preferably from 0.05to 5 mol/l, especially preferably from 0.05 to 1 mol/l.

[0095] The test solution may comprise salts of inorganic or organicacids. Examples are salts of citric acid, lactic acid or acetic acid oralso salts of phosphoric acid, hydrochloric acid and sulfuric acid, orcarbonate.

[0096] Preferably, the test solution comprises essentially 1,1- or1,2-salts. Examples of 1,1-salts are LiCl, NaCl, KCl and NH₄Cl. Examplesof 1,2-salts are CaCl₂, MgCl₂ and K₂SO₄ as described, for example, in G.Kortüm, Lehrbuch der Elektrochemie, 4th edition, Verlag Chemie 1966,page 156.

[0097] Preferably, the test solution consists partly or wholly of a bodyfluid. Especially preferably it consists partly or wholly of blood.

[0098] The process can be performed as a single measurement or can beperformed continuously.

[0099] The invention relates also to the use of an optical sensordescribed above for the ionic strength-independent optical determinationof the pH value of an aqueous test solution according to thefluorescence method.

[0100] The following Examples illustrate the invention.

EXAMPLE A Preparation of the Functionalised Fluorescent Dyes Example A1Preparation of 4-acryloylamidofluorescein (101)

[0101]

[0102] 5 g of 4-aminofluorescein are suspended in 200 ml of acetone and,at 0° C., 1.4 ml of acryloyl chloride in 2 ml of acetone are addeddropwise in the course of 10 min. The suspension is stirred for 3 hoursat room temperature. The crystals are filtered off, washed with acetoneand ether and dried. 5.7 g of compound (101) having a melting point of >200° C., are obtained. MS-FD: 402.

Example A2 Preparation of Compound (102)

[0103] a)

[0104] 29 ml of triethylene glycol monochlorohydrin and 20 g of sodiumazide are stirred overnight at 110° C. without solvent. The reactionmixture is diluted with ether and filtered off. The solvent isevaporated and the filtrate is concentrated under a high vacuum at110-115° C. Compound 103 is obtained in a yield of 86%.

[0105] b)

[0106] 8.2 g of NaH (washed with pentane) are suspended in 150 ml of drytetrahydrofuran. 30 g of compound (103) are added dropwise at 5° C. Themixture is stirred for a further 30 min. and subsequently reacted with38 ml of α-bromoacetic acid tert-butyl ester in 60 ml oftetrahydrofuran. The mixture is stirred overnight, the ether isevaporated and the organic phase is washed three times with water andonce with salt solution and then dried. The oil which remains isdistilled under a high vacuum at from 140 to 150° C. Compound (104) isobtained. FAB-MS: 290 [M+H]⁺. ¹H-NMR (CDCl₃): 1.45 ppm (9H, s, t-Bu);4.03 (2H, s, OCH₂COO).

[0107] c)

[0108] The azide group of compound (104) is quantitatively reduced withhydrogen, 5% Pd/C being used as catalyst and 1,4-dioxane being used assolvent. Compound (105) is obtained. ¹H-NMR (CDCl₃): 1.45 ppm (9H, s,t-Bu); 2.2 (2H, broad, NH₂); 2.9 (2H, t, J=6 Hz, CH₂N); 4.03 (2H, s,OCH₂COO).

[0109] d)

[0110] Compound (105) is dissolved in CH₂Cl₂ and treated with 1.5 equiv.of NEt₃ and 1.5 equiv. of acryloyl chloride at 0° C. The clear solutionis stirred for 5 hours, and then washed with water, salt solution andwater. The organic phase is dried. The oil which remains is purified bychromatography on silica gel with CH₂Cl₂ as eluant. The yield is 74% ofcompound (106). ¹H-NMR (CDCl₃): 1.45 ppm (9H, s, t-Bu); 4.03 (2H, s,OCH₂COO); 5.10 (1H, m, CH═C); 6.05-6.35 (2H, m, C═CH₂).

[0111] e)

[0112] The tert-butyl ester of compound (106) is removed with a 1:1mixture of trifluoroacetic acid and CH₂Cl₂ at room temperature in thecourse of 6 hours. The reaction product (107) is used directly for thenext step without being purified.

[0113] f) The acid (107) is treated with one equivalent ofcarbonyldiimidazole in tetrahydrofuran for 3 hours at room temperature.0.9 equivalent of 4-aminofluorescein in tetrahydrofuran is added to thatsolution and the mixture is stirred for 72 hours at room temperature.The reaction mixture is dried and the product is purified bychromatography on silica gel using MeOH/CH₂Cl₂ as eluant. Orangecrystals of compound (102) are obtained in a yield of 37%

[0114] with a melting point of 205° C. (decomposition). FAB-MS: 591[M+H]⁺, 613 [M+Na]⁺, 629 [M+K]⁺.

B) Preparation of the Copolymers Example B1

[0115] In an ampoule provided with a 3-way tap, which is connected to avacuum and nitrogen, 2.19 g (22.1 mmol) of N,N dimethylacrylamide, 2.81g (22.1 mmol) of N-tert-butylacrylamide, 200 mg of4-acryloylamidofluorescein (compound 101 from Example A1) and 25 mg ofazobisisobutyronitrile are dissolved in 15 ml of dimethyl sulfoxide. Theatmosphere in the ampoule is replaced with nitrogen by a freeze/thawcycle carried out three times. The ampoule is maintained at 60° C. for 2days in a water bath. The viscous contents of the ampoule are dilutedwith 100 ml of warm methanol and the copolymer is precipitated bycautiously pouring dropwise into 2 l of water with stirring. Thecopolymer is filtered, and roughly dried. The precipitation is repeatedtwice more. The thus purified end product is dried under a high vacuumat 60°.

[0116] Yield: 3.6 g or 69% of the theoretical yield, glass transitiontemperature T_(g)=156° C., content of N-tert-butylacrylamide=45.7% byweight (determined by IR-spectroscopy), inherent viscosity of a 0.5%solution in chloroform at 25° C. η_(inh)=1.07 dl/g.

Example B2

[0117] The procedure is as in Example B1 except that 125 mg of thefluorescent dye (101) from Example A1 are added. A copolymer having thefollowing characteristics is obtained. Yield 4.4 g or 86% of thetheoretical yield, T_(g)=152° C., content of N-tert-butylacrylamide=54.2% by weight (determined by IR-spectroscopy), inherent viscosity of a0.5% solution in chloroform at 25° C. η_(inh)=1.39 dl/g, dye contentdetermined by UV-spectroscopy= 2.2% by weight.

Example B3

[0118] The procedure is as in Example B1 except that 15 mg of thefluorescent dye (101) from Example A1 are added. A copolymer having thefollowing characteristics is obtained. Yield 3.4 g or 68% of thetheoretical yield, T_(g)=149° C., content of N-tert-butylacrylamide=58.7% by weight (determined by IR-spectroscopy), inherent viscosity of a0.5% solution in chloroform at 25° C. η_(inh)=1.68 dl/g, dye contentdetermined by UV-spectroscopy= 0.26% by weight.

Example B4

[0119] In an ampoule provided with a 3-way tap, which is connected to avacuum and nitrogen, 2.19 g (22.1 mmol) of N,N-dimethylacrylamide, 2.81g (22.1 mmol) of N-tert-butylacrylamide, 35 mg of compound 102 fromExample A2 and 25 mg of azobisisobutyronitrile are dissolved in 15 ml ofdimethyl sulfoxide. The atmosphere in the ampoule is replaced withnitrogen by a freeze/thaw cycle carried out three times. The ampoule ismaintained at 60° C. for 5 days in a water bath. The viscous contents ofthe ampoule are diluted with 25 ml of warm methanol and the copolymer isprecipitated by cautiously pouring dropwise into 1.5 l of ether withstirring. The copolymer is filtered, and roughly dried. Theprecipitation is repeated twice more. The thus purified end product isdried under a high vacuum at 50° for 2 days.

[0120] Yield: 4.05 g or 81% of the theoretical yield, glass transitiontemperature T_(g)=151° C., content of N-tert-butylacrylamide=51.9% byweight (determined by IR-spectroscopy), inherent viscosity of a 0.5%solution in tetrahydrofuran at 25° C. η_(inh)=1.34 dl/g, dye contentdetermined by UV-spectroscopy=0.44%.

Example B5

[0121] The procedure is as in Example B4 except that 2.31 g (23.29 mmol)of N,N-dimethylacrylamide, 2.69 g (19.06 mmol) ofN-methyl-N-butylacrylamide and 35 mg of the fluorescent dye (101) fromExample A1 are added.

[0122] A copolymer having the following characteristics is obtained.Yield 3.72 g or 74% of the theoretical yield, T_(g)=90° C., content ofN,N-dimethylacrylamide=55.7 mol % (determined by IR-spectroscopy),inherent viscosity of a 0.5% solution in chloroform at 25° C.η_(inh)=1.17 dl/g, dye content determined by UV-spectroscopy=0.62% byweight.

C Production of the Sensors Example C1

[0123] Glass substrates (platelets of 18 mm diameter) are first of allcleaned in 30% sodium hydroxide solution and then activated in 65%nitric acid. The activated platelets are then silanised with3-aminopropyltrimethoxysilane. The silanised platelets are left to reactfor 1 hour at room temperature in a solution ofO-(N-succinimidyl)-6-(4′-azido-2′-nitrophenylamino)-hexanoate indimethylformamide/borax buffer (5:1). The polymer of Example B1 (5%) isdissolved in methanol at from 20° to 25° and applied in the form of athin film, by spin-coating at a speed of 500 revs/min for 20 seconds,onto the platelets functionalised with azido groups, irradiated for 15min. and then dried for 12 h at 60° under nitrogen. The layerthicknesses of the membranes are approximately 1 μm.

Examples C2 to C5

[0124] Procedure is as in Example C1 and the corresponding polymers ofExamples B2 to B5 are used

D Application Examples

[0125] General Method

[0126] The sensors are mounted in a flow cell. The calibration andsample solutions are metered by pumps and conveyed through the cell. Themeasuring arrangement is thermostatically controlled. The light of ahalogen lamp (white light, excitation wavelength 480 nm) is conductedthrough an excitation filter and reflected at a dichroic mirror andfocussed by lenses onto the planar sensors. The fluorescent lightemitted by the sensors (at 520 nm) is collected by the same lens systemand directed by an emission filter and the dichroic mirror to aphotodiode. The fluorescence of the sensors is recorded while they arebeing acted upon by the calibration and sample solutions. The pH valuecan be determined directly from the measured value.

[0127] The following Table 1 illustrates the dependence of the pH on theionic strength of the electrolyte in the pH range of from 6.7 to 8.0that results on the basis of the different membrane compositions. TABLE1 pK_(a) Ionic strength Sensor from at ionic strength dependence at 0.1Example Amount of dye 0.1 mol/l and 0.3 mol/l C1 Comparison   4% by wt.not determinable very high (>1)¹ test. C2 Comparison  2.2% by wt. 7.3high (1)¹ test C3 0.26% by wt. 7.3 independent (0)¹ C4 0.44% by wt. 7.3independent (0)¹ C5 0.62% by wt. 7.5 independent (0)¹

What is claimed is:
 1. A water-insoluble copolymer which is composed ofa) from 39.9 to 60% by weight of N,N-dimethylacrylamide or N,N-dimethylmethacrylamide; b) from 60 to 39.9% by weight of a monomer of formula Iaor Ib

 wherein R_(a) is hydrogen or C₁-C₆alkyl and R_(b) is C₁-C₁₂alkyl; withthe proviso that R_(a) and Rb are not both methyl; c) from 0.1 to 0.7%by weight of a proton-sensitive fluorophore which is covalently bondedto the polymer; and d) from 0 to 20% by weight of a diolefiniccrosslinking component, the sum of the percentage weights of a) to d)being 100%.
 2. A water-insoluble copolymer according to claim 1 ,wherein N,N-dimethylacryl amide is used as monomer a).
 3. Awater-insoluble copolymer according to claim 1 , wherein R_(a) ishydrogen and R_(b) is a branched C₃-C₈alkyl.
 4. A water-insolublecopolymer according to claim 3 , wherein R_(a) is hydrogen, R_(b) istert-butyl and the ratio of monomer a) to monomer b) is 50 parts byweight to 50 parts by weight.
 5. A water-insoluble copolymer accordingto claim 1 , wherein R_(a) is methyl or ethyl and R_(b) is linearC₃-C₈alkyl.
 6. A water-insoluble copolymer according to claim 5 ,wherein R_(a) is methyl and R_(b) is n-butyl.
 7. A water-insolublecopolymer according to claim 1 , wherein the proton-sensitivefluorophore is selected from the group consisting of acridines,acridones, rhodamines, xanthenes, benzoxanthenes, pyrenes and coumarins.8. A water-insoluble copolymer according to claim 1 , wherein thefluorophore is covalently bonded to the polymer.
 9. A water-insolublecopolymer according to claim 1 , wherein the fluorophore is a compoundof formula II, III, IV, V or VI

wherein R₁, R₂, R₅ and R₆ are each independently of the others hydrogen,—SO₂—(C₁-C₆)alkylphenyl, C₁-C₃₀alkyl, C₁-C₃₀alkyl—CO— or a radical ofthe formula —(C_(n)H_(2n)—O—)_(m)—R₈; R₃ is hydrogen or—SO₂—(C₁-C₆)alkylphenyl; R₄ and R₇ are a C₁-C₃₀alkylene or a radical ofthe formula —(C_(n)H_(2n)—O—)_(m)—R₈; Z is a divalent radical —NH—CO—;R₈ is a direct bond or C₁-C₁₂alkylene; n is an integer from 2 to 6 and mis an integer from 1 to 10, with the proviso that the total number ofcarbon atoms is no more than 30; R₉ and R₁₀ are each independently ofthe other H, C₁-C₄alkyl, C₁-C₄alkoxy, C₁-C₄alkoxycarbonyl,C₁-C₄alkyl-SO₂— or halogen, and either R₁₁ is H and R₁₂ is a divalentradical —NH—CO—, —CO—NH—(C₂-C₁₂alkylene-O)—CO—, — CO—NH—(C₂-C₁₂alkylene—NH)—CO— or —C(O)—NH—(CH₂CH₂—O)_(1 to 6)—CH₂C(O)— NH—, orR₁₁ is a divalent radical —NH—CO—, —CO—NH—(C₂-C₁₂alkylene-O)—CO—,—CO—NH—(C₂-C₁₂alkylene-NH)— CO— or—C(O)—NH—(CH₂CH₂—O)_(1 to 6)—CH₂C(O)—NH—, and R₁₂ is H; or whereineither R₁₃ is H and R₁₄ is a divalent radical —NH—C(O)—,—CO—NH—(C₂C₁₂alkylene-O)—CO—, — CO—NH— (C₂-C₁₂alkylene—NH)—CO— or—C(O)—NH—(CH₂CH₂—O)_(1 to 6)—CH₂C(O)— NH—, or R₁₃ is a divalent radical—NH—C(O)—, —CO—NH—(C₂-C₁₂alkylene-O)—CO—, —CO—NH—(C₂-C₁₂alkylene-NH)—CO— or —C(O)—NH—(CH₂CH₂—O)_(1 to 6)—CH₂C(O)—NH—, and R₁₄ is H, whereinthe radical — COOH is each in free form or in salt form, or aC₁-C₂₀alkyl ester thereof.
 10. A water-insoluble copolymer according toclaim 9 , wherein R₁ and R₂ of the fluorophore of formula II are eachindependently of the other hydrogen or linear C₁₂-C₂₄alkyl.
 11. Awater-insoluble copolymer according to claim 9 , wherein R₄ of thefluorophore of formula II is a linear C₂-C₁₆alkylene or a radical of theformula —(C₂H₄—O—)_(m)—R₈ wherein R₈ and m are as defined in claim 9 .12. A water-insoluble copolymer according to claim 9 , wherein R₅ and R₆of the fluorophore of formula III are each independently of the otherlinear C₂-C₁₂alkyl.
 13. An optical sensor for an ionicstrength-independent pH value determination, consisting of A) atransparent support material B) a layer of water-insoluble copolymersthat are composed of a) from 39.9 to 60% by weight ofN,N-dimethylacrylamide or N,N-dimethylmethacrylamide; b) from 60 to39.9% by weight of a monomer of formula Ia or Ib

 wherein R_(a) is hydrogen or C₁-C₆alkyl and R_(b) is C₁-C₁₂alkyl; withthe proviso that R_(a) and Rb are not both methyl; c) from 0.1 to 0.7%by weight of a proton-sensitive fluorophore which is covalently bondedto the polymer; and d) from 0 to 20% by weight of a diolefiniccrosslinking component, the sum of the percentage weights of a) to d)being 100%.
 14. A sensor according to claim 13 , wherein the fluorophoreis selected from the group consisting of acridines, acridones,rhodamines, xanthenes and benzoxanthenes, pyrenes, coumarins andfluoresceins.
 15. A sensor according to claim 13 , wherein the thicknessof the polymer layer B) is from 0.1 to 500 μm.
 16. A process for theionic strength-independent, reversible optical determination of the pHvalue of an aqueous sample according to the fluorescence method, inwhich an optical sensor, consisting of A) a transparent support materialB) a layer of water-insoluble copolymers that are composed of a) from39.9 to 60% by weight of N,N-dimethylacrylamide or N,N-dimethylmethacrylamide; b) from 60 to 39.9% by weight of a monomer of formula Iaor Ib

 wherein R_(a) is hydrogen or C₁-C₆alkyl and R_(b) is C₁-C₁₂alkyl; withthe proviso that R_(a) and Rb are not both methyl; c) from 0.1 to 0.7%by weight of a proton-sensitive fluorophore which is covalently bondedto the polymer; and d) from 0 to 20% by weight of a diolefiniccrosslinking component, the sum of the percentage weights of a) to d)being 100%, is brought into contact with an aqueous test sample andirradiated with excitation light, the fluorescence is measured, and thepH value is calculated from the measured fluorescence intensity takingcalibration curves into consideration.
 17. A process according to claim16 , wherein the test solution has a pH of from 6.5 to 8.5.
 18. Aprocess according to claim 16 , wherein the ionic strength of the testsolution is from 0.05 to 5 mol/l.
 19. A process according to claim 16 ,wherein the ionic strength is provided essentially by 1,1- or 1,2-salts.20. A process according to claim 16 , wherein the test solution consistspartly or wholly of a body fluid.
 21. The use of an optical sensoraccording to claim 13 for the ionic strength-independent determinationof the pH value of an aqueous test solution according to thefluorescence method.